Conventional Cytogenetics In Detecting Recurring Chromosomal Abnormalities In Hematopoietic Disorders

Conventional cytogenetic analysis using G-banding is a powerful technique that enables one to gain a complete picture of the human genome at a glance (Fig. 2). Bone marrow is the tissue of choice for conventional cytoge-netic studies of acquired chromosomal abnormalities in most hematological conditions. Nevertheless, a stimulated culture of peripheral blood is sometimes performed besides an unstimulated culture of the bone marrow to ascertain whether an observed chromosomal abnormality, when present in all sampled cells, is constitutional in origin.[13,14]

The first consistent acquired chromosomal abnormality that was associated with a single cancer type, CML, was described by Nowell and Hungerford.[15] The marker chromosome, named the Philadelphia (Ph1) chromosome, in honor of the city where it was discovered, was later found by Rowley[16] to be a reciprocal translocation between chromosomes 9 and 22 using the G-banding technique. This translocation, in which the c-abl oncogene

Fig. 2 A normal G-banded male diploid karyotype. [Courtesy of Cytogenetics Department (Dr. H.F.L. Mark, Director), Presbyterian Laboratory Services, Charlotte, North Carolina.]

on 9q34 is fused to the breakpoint cluster region (bcr) locus on chromosome 22, results in a hybrid gene. Subsequently, this gene produces a fusion protein with increased tyrosine kinase activity and eventually leads to myeloid cell transformation. Figure 3 is a simplified illustration of the structural chromosomal rearrangement giving rise to the Philadelphia translocation. It is given special attention here because this reciprocal translocation mechanism was subsequently found to be a common theme: Many of the other structural chromosomal

Fig. 3 A simplified illustration of the structural chromosomal rearrangement giving rise to the Philadelphia translocation. Der(22) denotes the Philadelphia chromosome. Chromosomes are not necessarily drawn to scale.

abnormalities found in acute myelocytic leukemia can be explained by the same or similar paradigm. Because of space limitations, the detailed breakage and reunion events leading to the common translocations encountered in the various AML subtypes will not be repeated here. Instead, the reader is referred to the figures provided in the products (probes) section of the Vysis website,[17] as well as in a review by Anastasi and Roulston.[18] Selected examples of the common chromosomal translocations encountered in AML are given in Fig. 1.

Many patients with AML have characteristic recurring chromosomal abnormalities that were detectable in metaphase preparations by conventional cytogenetics, even before the advent of fluorescent (or fluorescence) in situ hybridization (FISH). FISH is a powerful technique for detecting both numerical and structural cytogenetic abnormalities in nondividing (interphase) as well as dividing (metaphase) cells. On the other hand, conventional cytogenetics can only be performed when the mitotic index is within acceptable limits (i.e., when there is a sufficient number of metaphases to complete a meaningful analysis). Another advantage of FISH is that a large number of cells can readily be scored using the technique, whereas metaphase cytogenetic analysis is cumbersome and labor intensive when analyzing beyond the standard number of cells.[13,19-22]


FISH is a molecular cytogenetic technique that enables the detection of a probe that binds to homologous sequences on a chromosome that is fixed on a glass slide. FISH can be performed on a variety of specimen types such as peripheral blood, bone marrow, and pathological sections.1-23,24-1 Probes used for FISH for cancer applications are most commonly locus-specific DNA probes or alpha-satellite centromere enumeration probes, or a combination of both. The different types of FISH probes and their applications are extensively discussed elsewhere.[25-27]

The technical steps involved in performing FISH are similar to nonfluorescent in situ hybridization techniques. Figure 4 depicts a simplified FISH procedure used in many clinical and research cytogenetic laboratories, whereas Fig. 5 is a simplified schematic representation of the detection of the Philadelphia translocation using FISH. This translocation, characteristic of CML, is used here as an example to illustrate the utility of FISH. Variations of the same basic paradigm enable the detection of most structural chromosomal rearrangements commonly found in AML and other hematopoietic disorders. Other details associated with the principles and techniques of FISH were described by Blancato and Haddad.[25] Research and clinical applications of FISH in cancer cytogenetics include detection of numerical chromosomal abnormalities (aneuploidies), structural chromosomal abnormalities such as translocations, inversions, deletions, duplications, isochromosomes, derivative chromosomes, dicentrics, rings, acentrics, double minutes, amplifications and marker chromosome identification.1-28-30-1 FISH is especially useful in studying a large number of non-dividing cells for the determination of clonality, particularly in suboptimal cancer preparations where the mitotic index is low.[31] Additional applications have been summarized elsewhere.[27] Examples of clinical applications of FISH to detect chromosomal abnormalities in AML are further discussed below.

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